title: An insight into the hepatocellular death induced by amphetamines, individually and in combination: the involvement of necrosis and apoptosis.
abstract: The liver is a vulnerable target for amphetamine toxicity, but the mechanisms involved in the drug's hepatotoxicity remain poorly understood. The purpose of the current research was to characterize the mode of death elicited by four amphetamines and to evaluate whether their combination triggered similar mechanisms in immortalized human HepG2 cells. The obtained data revealed a time- and temperature-dependent mortality of HepG2 cells exposed to 3,4-methylenedioxymethamphetamine (MDMA, ecstasy; 1.3 mM), methamphetamine (3 mM), 4-methylthioamphetamine (0.5 mM) and D-amphetamine (1.7 mM), alone or combined (1.6 mM mixture). At physiological temperature (37 °C), 24-h exposures caused HepG2 death preferentially by apoptosis, while a rise to 40.5 °C favoured necrosis. ATP levels remained unaltered when the drugs where tested at normothermia, but incubation at 40.5 °C provoked marked ATP depletion for all treatments. Further investigations on the apoptotic mechanisms triggered by the drugs (alone or combined) showed a decline in BCL-2 and BCL- XL mRNA levels, with concurrent upregulation of BAX, BIM, PUMA and BID genes. Elevation of Bax, cleaved Bid, Puma, Bak and Bim protein levels was also seen. To the best of our knowledge, Puma, Bim and Bak have never been linked with the toxicity induced by amphetamines. Time-dependent caspase-3/-7 activation, but not mitochondrial membrane potential (∆ψm) disruption, also mediated amphetamine-induced apoptosis. The cell dismantling was confirmed by poly(ADP-ribose)polymerase proteolysis. Overall, for all evaluated parameters, no relevant differences were detected between individual amphetamines and the mixture (all tested at equieffective cytotoxic concentrations), suggesting that the mode of action of the amphetamines in combination does not deviate from the mode of action of the drugs individually, when eliciting HepG2 cell death.
authors.affiliation: Faculdade de Medicina, Universidade do Porto, 4200-319, Porto, Portugal, diana.dds@gmail.com.
authors.forename: Diana
authors.initials: D
authors.lastname: Dias da Silva
authors.forename: Helena
authors.initials: H
authors.lastname: Carmo
authors.forename: Adam
authors.initials: A
authors.lastname: Lynch
authors.forename: Elisabete
authors.initials: E
authors.lastname: Silva
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000662:Amphetamines / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D000697:Central Nervous System Stimulants / Q000633:toxicity*; D056486:Chemical and Drug Induced Liver Injury / Q000473:pathology*; D004734:Energy Metabolism / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D008566:Membranes / Q000187:drug effects / Q000648:ultrastructure; D008928:Mitochondria / Q000187:drug effects; D009336:Necrosis; D009499:Neutral Red; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D048348:Reverse Transcription / Q000187:drug effects; D013778:Tetrazolium Salts; D013844:Thiazoles
chemical_list: D000662:Amphetamines; D000697:Central Nervous System Stimulants; D013778:Tetrazolium Salts; D013844:Thiazoles; D009499:Neutral Red; D012313:RNA; D000255:Adenosine Triphosphate; D053148:Caspase 3; D053179:Caspase 7; C022616:thiazolyl blue